Abstract The pathological hallmarks of Alzheimer?s disease are the neurofibrillary tangles and amyloid plaques that form in gray matter regions of the cerebral cortex. Misfolded and aggregated proteins reside within those pathological features, which may to be central to the neuronal death that causes regional atrophy, dementia, and ultimately death. The cerebrospinal fluid that bathes the central nervous system is the fluid most proximal to the disease and has been the focus of biomarker discovery for more than 30 years. There are two important gene products that are present in cerebrospinal fluid and have been shown to be predictive of disease activity and cognitive decline in patients that present with mild cognitive impairment: (1) the amyloid precursor protein, which gives rise to A?(1-42) and other fragments, and (2) tau, which has many phosphorylation sites. The deposition of A?(1-42) as amyloid aggregates appears to lead to the death of adjacent neurons. The resulting injury increases the amount of tau protein released into the cerebrospinal fluid. From previous biomarker studies of A? proteins and tau, it seems likely that the entire disease process, from A?(1-42) deposition to cell death, is orchestrated differently than in other neurodegenerative diseases. As a result, it is a compelling hypothesis that novel biomarkers, which may be mediators of disease, are present in the cerebrospinal fluid and could add more information over the current biomarkers used in the diagnosis of Alzheimer?s disease. There have been previous attempts to use discovery proteomics to identify proteins that are differentially expressed in cerebrospinal fluid in patients with Alzheimer?s disease compared with controls and there have been few efforts to expand upon these studies in a clinically meaningful way. However, some of these efforts have identified the presence of additional fragments of amyloid precursor protein [i.e., in addition to A?(1-42)] and other proteins, which suggests that post-translational changes may be important in the formation of amyloid, the dysfunction of neuronal synapses, and the subsequent death of neurons. In this project entitled, ?Identification of mass spectrometric targets and development of multiplex assays for disease management,? our objective is to identify the post-translational modifications that lead to altered LP/EV concentration, molecular weight, and stability in cerebrospinal fluid. Part of the reason that the clinical translation of A?(1-42), tau, and phosphorylated tau as cerebrospinal fluid biomarkers took decades was that reproducible assays were not available early on. As a result, another overarching goal of this project is to develop precise, transferable, validated targeted proteomic assays to quantify proteins in cerebrospinal fluid that can be used to investigate disease mechanism and predict poor outcomes.